Gene therapy and modulation of gene expression holds enormous potential for a new era in human medicine. These methodologies will allow treatment for conditions that heretofore have not been addressable by standard medical practice.
Recombinant transcription factors comprising the DNA binding domains from zinc finger proteins (“ZFPs”) or TAL-effector domains (“TALEs”) have the ability to regulate gene expression of endogenous genes (see, e.g., U.S. Pat. Nos. 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054). Clinical trials using these engineered transcription factors containing zinc finger proteins have shown that these novel transcription factors are capable of treating various conditions. (see, e.g., Yu et al. (2006) FASEB J. 20:479-481).
Another area of gene therapy that is especially promising is the ability to genetically engineer a cell to cause that cell to express a product not previously being produced in that cell. Examples of uses of this technology include the insertion of a gene encoding a novel therapeutic protein, insertion of a coding sequence encoding a protein that is lacking in the cell or in the individual, insertion of a wild type gene in a cell containing a mutated gene sequence, and insertion of a sequence that encodes a structural nucleic acid such as a microRNA or siRNA.
Transgenes can be delivered to a cell by a variety of ways such that the transgene becomes integrated into the cell's own genome and is maintained there. In recent years, a strategy for transgene integration has been developed that uses cleavage with site-specific nucleases for targeted insertion into a chosen genomic locus. Nucleases specific for targeted genes (including “safe harbor” loci such as CCR5, CXCR4, AAVS1, albumin or Rosa) can be utilized such that the transgene construct is inserted by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes. See, for example, U.S. Pat. Nos. 8,623,618; 8,034,598; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983; 20130177960 and 20150056705, the disclosures of which are incorporated by reference in their entireties for all purposes. Nuclease-mediated integration offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches that rely on random integration of the transgene, since it allows exact transgene positioning for a minimal risk of gene silencing or activation of nearby oncogenes.
Engineered nucleases, including zinc finger nucleases, TALENs, CRISPR/Cas nuclease systems, and homing endonucleases designed to specifically bind to target DNA sites are useful in genome engineering and gene therapy. For example, zinc finger nucleases (ZFNs) and TALENs (including TALENs comprising Fok1-TALE DNA binding domain fusions, Mega TALs and cTALENs) are proteins comprising engineered site-specific zinc fingers or TAL-effector domains fused to a nuclease domain. Such nucleases have been successfully used for genome modification in a variety of different species at a variety of genomic locations. Additionally, clinical trials using engineered zinc finger nucleases have also demonstrated therapeutic utility (see, e.g. Tebas et at (2014) New Eng J Med 370(10):901).
These approaches have great potential for the treatment of diseases where the targeted gene product is considered un-druggable by small molecule approaches for a variety of reasons such as inaccessibility of the gene product or similarity with other essential gene products. Thus, there remains a need for the development of targeted fusion molecules (e.g. nucleases and transcription factors) for the prevention or treatment of diseases associated with the expression of disease-causing gene products.